KR20070050993A - Peptide originating in hepatitis c virus - Google Patents

Abstract

A hepatitis C virus derived peptide is disclosed which comprises an HLA binding motif in a sequence and is also recognized by an antibody detected in a hepatitis C virus (HCV) patient. The peptides of the present invention are useful for the diagnosis of hepatitis C virus, for the treatment of hepatitis C, and for the prognosis.

HLA binding motif

Description

Peptide derived from hepatitis C virus {PEPTIDE ORIGINATING IN HEPATITIS C VIRUS}

1 is a graph showing blood IgG measurement results of HCV-infected patients by ELISA.

Fig. 2 is a graph showing blood IgG measurement results of patients with HCV infection by ELISA in the present invention.

FIG. 3 is a graph showing the results of absorption and elution experiments of representative examples of C-35 peptide reactive antibodies in the patient serum shown in FIG. 1 according to the present invention.

Fig. 4 is a graph showing the results of CTL induction of six HLA-A24 binding peptides (No 3, 12, 13, 25, 32, 38) derived from HCV in the present invention.

Fig. 5 is a graph showing the results of CTL induction of HLA-A2 binding peptides (No 40 to No 57) derived from HCV in the present invention.

6 is a graph showing representative results of cytotoxic activity of peptide-stimulated PBMCs by the ⁵¹ Cr glass test in the present invention.

7 is a graph showing representative results of cytotoxic activity of peptide-stimulated PBMCs by the ⁵¹ Cr glass test in the present invention.

8 is a graph confirming HLA class I restriction of cytotoxicity of peptide-stimulated PBMCs by inhibition test using anti-CD8 monoclonal antibody in the present invention.

9 is a graph showing detection of anti-peptide IgG in HCV infected patient serum.

10 is a graph showing detection of anti-peptide IgG in HCV infected patient serum.

11 is a graph showing the results of an adsorption test for examining the specificity of anti-peptide IgG in serum of HCV infected patients.

12 is a graph showing the results of an elution test for examining the specificity of anti-peptide IgG in serum of HCV infected patients.

Figure 13 is a graph showing IFN-γ production by peptide stimulation PBMCs.

14 is a graph showing IFN-γ production by peptide stimulation PBMCs.

15 is a graph showing cytotoxicity of peptide stimulated PBMCs.

16 is a graph showing cytotoxicity of peptide stimulated PBMCs.

FIG. 17 is a graph showing CTL activity of peptide stimulated PBMCs on cells expressing NS5A. FIG.

FIG. 18 is a graph showing CTL activity of peptide stimulated PBMCs on cells expressing NS5A. FIG.

FIG. 19 is a graph showing CTL activity of peptide stimulated PBMCs on cells expressing NS5A.

20 is a graph showing the specificity of anti NS5A-2132 IgG activity.

Fig. 21 is a graph showing the analysis results of cell proliferation inhibition by anti-NS5A-2132 IgG.

Fig. 22 is a graph showing the analysis results of ADCC activity of anti-NS5A-2132 IgG.

23 is a graph showing detection of anti-C-35 antibody and anti-NS5A-2132 antibody in various diseases.

24 is a graph showing HLA or HCV genotype restriction of anti-peptide antibodies.

25 is a graph showing HLA or HCV genotype restriction of anti-peptide antibodies.

It is a graph which shows the measurement result of anti-NS5A-2132 antibody in patient serum.

Fig. 27 is a graph showing the results of measurement of anti-NS5A-2132 IgG in patient serum.

28 is a graph showing the levels of anti-NS5A antibodies and HCV RNA levels in patient serum.

29 is a graph showing detection of anti-C-35 antibody and anti-NS5A-2132 antibody in a cohort study.

30 is a graft showing detection of anti-NS5A-2132 antibody in cohort study.

Fig. 31 is a graph showing blood IgG measurement results of patients with HCV infection by Luminex method in the present invention.

Fig. 32 is a graph showing blood IgG measurement results of patients with HCV infection by Luminex method in the present invention.

Figure 33 shows the course of treatment of hepatitis C using the peptide of the present invention.

Figure 34 shows the course of treatment of hepatitis C using the peptide of the present invention.

The present invention relates to hepatitis C virus (HCV) peptides useful for the diagnosis of hepatitis C virus infection and for the treatment of hepatitis C.

Also specifically, the present invention relates to HCV peptides, polypeptides containing HCV peptides, HCV peptides or nucleotides encoding such polypeptides, or antibodies or antibody-like active substances that recognize them, vectors containing said nucleotides, HCV peptides or A method of inducing cytotoxic T cells using a polypeptide, or a method for detecting hepatitis C virus using an HCV peptide, the polypeptide, the nucleotide, or the antibody-antibody-like active substance, diagnosis, prevention, or The present invention relates to a method of treatment, a method of predicting prognosis, and a pharmaceutical composition for treating hepatitis C containing them.

Viral hepatitis includes hepatitis A, which is mainly infected with the virus, and hepatitis B, which is mainly infected by blood transfusion, in addition to hepatitis B, which is infected through the blood. The hepatitis C is one of the diseases with a high rate of chronic and transition to cirrhosis or liver cancer.

Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flavivirus family, and it is known that there are more than 2 million hepatitis C virus infections in Japan, and more than 170 million people worldwide.

Although the combination of interferon and ribavirin is performed for this hepatitis C, it is effective only for patients between 30% and 40%, and there is no current effective therapy for most patients. Thus, there is a strong social call for urgently developing safe, effective and simple yet low cost diagnostic and treatment tools.

There is evidence indicating that both cellular and humoral immune responses play a major role in suppressing HCV infection (WO89 / 04669). Over the past decade, a number of studies have been conducted to find high immunogenic HCV peptides and numerous HCV peptides have been found that can induce cellular or humoral immune responses (Hunziker et al., Mol Immunol. 2001 Dec; 38 (6): 475-84). However, none of these peptides are clinically effective, and as a result, there is no means for immunotherapy that is prophylactically or therapeutically effective at present. This is presumably due to the weak immunogenicity of these peptides.

In addition, the present inventors have shown that the antigenic peptides recognized by some cytotoxic T lymphocytes (CTLs) are capable of eliciting both cellular and humoral immune responses both in vitro and in vivo. Has already reported (Gohara et al., J Immunother. 2002 Sep-Oct; 25 (5): 439-44, Mine et al., Clin Cancer Res. 2001 Dec; 7 (12): 3950-62 , Tanaka et al, J Immunother. 2003 Jul-Aug; 26 (4): 357-66, Mine et al., Cancer Sci. 2003 Jun; 94 (6): 548-56).

It is also contemplated that HCV peptides recognized by both cellular and humoral immune responses are more immunogenic than those recognized only by cellular immune responses.

As a new attempt to meet this social need, a series of studies show that HCV peptides capable of eliciting both cellular and humoral immune responses have higher immunogenicity than peptides with only humoral immune responses. Since knowledge has been accumulated, there are attempts to specify such peptides.

Thus, identifying such peptides and investigating whether they would be candidates as novel therapeutic and diagnostic tools for inhibiting HCV infection is considered beneficial.

Therefore, the present inventors investigated whether IgGs reactive with peptides recognized by HLA-A2 and HLA-A24 constrained cytotoxic T lymphocytes (CTL) can be detected from serum of HCV-infected patients. It was found that IgG specific for epitopes was detected in the majority of patients with HCV infection, regardless of the different HLA class I type or the different HCV geno type. In addition, from this result, it has been found that such a peptide can provide a novel tool for the prevention and treatment based on the peptide, thus completing the present invention.

Accordingly, an object of the present invention is to provide an HCV peptide which is recognized by a cellular immune response and a humoral immune response and which is highly immunogenic.

Disclosure of the Invention

In order to solve the said subject, this invention provides the hepatitis C virus derived peptide which contains the HLA binding motif in a sequence, and is recognized by the blood antibody of a hepatitis C virus infection patient.

In a preferred embodiment of the present invention, a hepatitis C virus-derived peptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, 16, 20, and 38 in the sequence table, and at least 70% homology with the amino acid sequence of the peptide Hepatitis C virus-derived peptides having an amino acid sequence having, and Hepatitis C virus-derived peptides further having recognition by HLA-A2 or HLA-A24 constrained cytotoxic T cells.

Another aspect of the invention provides a polypeptide containing the hepatitis C virus derived peptide. This aspect of the invention provides, as a preferred embodiment, a polypeptide having an amino acid sequence having at least 70% homology with the amino acid sequence of the polypeptide and recognition by HLA-A2 or HLA-A24 constrained cytotoxic T cells. It provides a polypeptide having.

In addition, another aspect of the present invention provides a nucleotide encoding the hepatitis C virus-derived peptide or polypeptide thereof.

Another form of the invention provides antibodies and antibody-like active substances that can immunologically recognize the hepatitis C virus-derived peptide or polypeptide thereof.

In another aspect, the present invention provides a vector containing the nucleotide.

Still another aspect of the present invention provides a method for inducing cytotoxic T cells using the hepatitis C virus-derived peptide, the nucleotide encoding the polypeptide.

In another aspect, the present invention provides a method for detecting hepatitis C virus, using the hepatitis C virus-derived peptide, polypeptide, nucleotide, or an antibody or an antibody-like active substance.

In addition, another aspect of the present invention provides a method for diagnosing HCV infection related diseases such as hepatitis C, using the hepatitis C virus-derived peptide, polypeptide, nucleotide, or an antibody or antibody-like active substance.

In another aspect, the present invention provides a method for treating an HCV infection related disease such as hepatitis C, using the hepatitis C virus-derived peptide, polypeptide, nucleotide, or an antibody or an antibody-like active substance.

In still another aspect, the present invention provides a pharmaceutical composition containing the hepatitis C virus-derived peptide, polypeptide, nucleotide, or antibody or antibody-like active substance as an active ingredient. Moreover, as a preferable aspect, the pharmaceutical composition whose pharmaceutical composition is a hepatitis C virus vaccine is provided.

In another aspect, the present invention provides a method for predicting the prognosis of an HCV infection related disease such as hepatitis C, using the hepatitis C virus-derived peptide, polypeptide, nucleotide, or an antibody or an antibody-like active substance.

In still another aspect, there is provided a kit for diagnosing or predicting prognosis of hepatitis C, containing the hepatitis C virus-derived peptide, polypeptide, nucleotide, or antibody or antibody-like active substance.

Detailed description of the invention

The hepatitis C virus-derived peptide according to the present invention is an HCV peptide that contains an HLA binding motif in a sequence and is also recognized by blood antibodies of a hepatitis C virus infected patient.

In addition, the HCV peptide according to the present invention contains an HLA binding motif, that is, an HLA-A2 or HLA-A24 binding motif in its sequence. Moreover, this HCV peptide is recognized by cellular immune response and humoral immune response, and is characterized by high immunogenicity.

As such HCV peptide, the peptide of Table 1 and Table 3 is contained, for example. One particularly preferred HCV peptide in the present invention is a peptide having an HLA-A2 binding motif having the following amino acid sequence:

C-35: YLLPRRGPRL (SEQ ID NO: 1)

to be. In patients with HCV infection, the detection rate and HCV infectivity specificity of peptide-reactive IgG antibodies to this peptide are very high, 93% and 100%, respectively. Also particularly preferred in the present invention are other HCV peptides having HLA-A24 binding motifs having the following amino acid sequences:

NS5A-2132: RYAPACKPL (SEQ ID NO: 2)

E2-488: HYAPRPCGI (SEQ ID NO: 3)

E1-213: VYEAADMIM (SEQ ID NO: 4)

NS3-1081: VYHGAGSKTL (SEQ ID NO: 5)

C-176: IFLLALLSCL (SEQ ID NO: 6)

C-173: SFSIFLLALL (SEQ ID NO: 7)

EYVLLLFLL (SEQ ID NO: 8)

PYIEQGMQL (SEQ ID NO: 16)

IFTITKILL (SEQ ID NO: 20)

SFAIKWEYVL (SEQ ID NO: 38)

to be. In particular, NS5A-2132 can induce both cellular and humoral immunity in many patients with HLA-A24 positive.

The HCV peptide according to the present invention may be a peptide having at least 70%, preferably at least 80%, more preferably at least 90% homology with the amino acid sequence of the peptide.

In addition, the HCV peptide according to the present invention may further contain a peptide having recognition ability by HLA-A2 or HLA-A24 restrictive cytotoxic T cells (CTL). Antigen peptides capable of binding to HLA, wherein the antigenic protein produced in the cell is composed of peptides degraded within the cell, have a motif in the sequence for each type of HLA, and cytotoxic T cells (CTL) are expressed in this antigen peptide and HLA. Recognizes a complex of injured HCV infected cells.

The polypeptide according to the present invention contains the amino acid sequence of the peptide of the present invention as described above in the amino acid sequence, and the total number of the amino acids is not particularly limited. In the polypeptide of the present invention, the remaining amino acids exceeding the amino acids constituting the peptide are located on the N-terminal side and / or C-terminal side or both sides of the amino acid of the peptide. Thus, the polypeptide has substantially the same functions and functions as those of the peptide. In the present specification, even when only "peptide" is described, "polypeptide" shall also be included unless otherwise specified.

Peptides having an amino acid sequence determined as described above can be obtained by conventional chemical synthesis, enzymatic digestion of protein molecules, gene recombination techniques using a host transformed to express a base sequence encoding a desired amino acid sequence.

When the target peptide is produced by chemical synthesis, it can be produced by a method commonly known per se in ordinary peptide chemistry, and can be synthesized by solid phase synthesis using a peptide synthesizer, for example. have. The so-called peptide obtained in this way can be purified by a purification method usually used in protein chemistry, for example, salting out method, ultrafiltration method, reverse phase chromatography method, ion exchange chromatography method, affinity chromatography method and the like.

On the other hand, when the desired peptide is produced by genetic recombination technology, for example, a DNA fragment encoding the desired amino acid sequence synthesized as described above is inserted into a suitable expression vector, and the microorganism or animal cell is used using this expression vector. The desired peptide can be obtained by transforming and culturing the obtained transformant. As the expression vector that can be used, plasmids, viral vectors and the like which are known in the art can be used.

As a transformation method of the host cell using the expression vector in this peptide production technique, a method known per se, for example, calcium chloride method, calcium phosphate co-precipitation method, DEAE dextran method, lipofectin method, electroporation method, etc. Can be used and it is appropriate to select appropriately based on the host cell to be used. The obtained peptide can be purified by the above purification method from the cell extract or culture supernatant recovered from the cultured medium.

A nucleotide as another form of this invention contains the said hepatitis C virus derived peptide or the oligonucleotide or polynucleotide containing the said polypeptide, and contains ribonucleotide or deoxynucleotide. The nucleotide may be modified by a known method in the art. The nucleotide modification includes, for example, known labeling, methylation, "caps", substitution of one or more natural nucleotides by analog, intranucleotide modification, and the like. Intranucleotide modifications include, for example, nonionic alterations (eg, methylphosphate, phosphate triesters, phosphoramidates, etc.), ionic alterations (eg, phosphorothioates, phosphorodithioates, etc.). For example, modifications including pendant moieties such as proteins (eg, nucleases, toxins, antibodies, signal peptides, etc.), modifications by chelating agents (eg, metals, boron, etc.), and the like. have.

The nucleotide sequence makes it possible to provide peptides and polypeptide sequences of HCV antigens encoded in the genome of HCV, and can also provide useful peptides as components of diagnostic tests or vaccines.

Obtaining the nucleotides of the present invention enables the construction of nucleotide probes and peptides useful for diagnosing a disease associated with HCV infection, or useful for screening HCV infection in blood or in blood products. From this nucleotide sequence, for example, 24 to 30 nucleotides or longer DNA oligomers can be synthesized. This nucleotide can be used as a probe for detecting HCV RNA in the serum of a subject and also for screening for the presence of HCV in blood or blood products.

This nucleotide sequence also allows the design and production of HCV specific peptides that are also useful as reagents for diagnosing the presence of antibodies to HCV. In addition, the antibody against the purified peptide derived from this sequence can also be used for detecting HCV antigen in HCV infected persons and HCV infected blood products.

An antibody as one of the other forms of the present invention is a hepatitis C virus-derived peptide, a chimeric antibody, a modified antibody, a monovalent antibody, Fab, F (ab ') ₂ , Fab', single chain Fv that are reactive with the polypeptide. (scFv) proteins and single domain antibodies. In addition, the antibody can immunologically recognize the peptide or the polypeptide.

The vector as one of the other forms of this invention contains the nucleotide, and is a construct which can transform the selected host cell, and can express the heterologous code sequence in the host. This expression vector may be either a cloning vector or an insertion vector.

As a cloning vector, a plasmid, a virus (for example, an adenovirus vector), etc. are used. The cloning vector is also a replicon (eg, plasmid, chromosome, virus, cosmid, etc.) capable of transforming host cells and having the ability to replicate nucleotides within the cell.

On the other hand, the insertion vector does not function as a replicon in the host cell, but in order to stably transform the host, the insertion vector has the ability to insert a liquor into the replicon (typically chromosome) in the host. Is a vector.

As another aspect of the present invention, a method of inducing cytotoxic T cells targeting HCV cells by using the peptide is included. The method of induction of the present invention, for example, adds the peptide to cells expressing HLA-A2 and presents it on HLA-A2, stimulating T cells with the cells presented by HLA-A2. It is preferable that the T cells are induced by CTL. The HLA-A2 expressing cells used in this method may be obtained from blood of an HCV patient, but can also be prepared by introducing a gene encoding HLA-A2 into non-FHLA-A2 expressing cells. Therefore, the peptide according to the present invention can be used not only for the detection and diagnosis of HCV, but also as a pharmaceutical composition such as a vaccine for hepatitis C virus-related diseases.

In addition, since CTL thus induced targets HCV infected cells and attacks the HCV infected cells, it can be used as a pharmaceutical composition such as cell therapy as well as the peptide.

In addition, the hepatitis C virus-derived peptide, polypeptide, nucleotide and / or antibody-antibody-like active substance, which is another embodiment of the present invention, is useful for detecting and diagnosing HCV infection.

The detection of HCV and the diagnosis of HCV related diseases can be performed, for example, using the interaction and / or reactivity with the nucleic acid sequence encoding the peptide to detect the presence of the peptide, the detection of the corresponding amount of nucleic acid sequence present, The determination can be carried out by determining the biodistribution in the individual of the peptide and / or determining the amount of abundant in the specimen derived from the individual. In short, the detection of HCV and the diagnosis of HCV-related diseases can be performed by assaying the peptide as a marker. In addition, the measurement can be performed by a measurement method known per se, and examples of such a measurement method include a method using an antigen antibody reaction system, an enzyme reaction system, a PCR reaction system, and the like.

Confirmation of the therapeutic effect and prediction of the prognosis of HCV related disease can be performed by monitoring the blood concentration of the antibody which has reactivity with the peptide in the subject with HCV related disease. Particularly preferably, the level of anti C-35 IgG or anti NS5A-2132 IgG in the blood of the subject is measured.

As a pharmaceutical composition which is another form of this invention, a vaccine is mentioned, for example. The vaccine consists of hepatitis C virus-derived peptides, polypeptides and / or nucleotides, and may contain a pharmaceutically acceptable adjuvant and / or carrier as appropriate. As an adjuvant, an adjuvant capable of enhancing an immune response, for example, Freund's incomplete adjuvant, aluminum hydroxide gel and the like can be used. As the carrier, for example, PBS, diluents such as distilled water, physiological saline and the like can be used.

The pharmaceutical composition of the present invention can be administered by parenteral or transdermal route, such as oral, intravenous or subcutaneous administration, depending on the form of use. Examples of the formulation include tablets, granules, soft capsules, hard capsules, liquids, emulsions, emulsifiers, and the like. Although the dosage of such a pharmaceutical composition can vary suitably according to the symptoms, etc. of the patient to administer, In general, it is preferable that it is 0.1-10 mg as the peptide amount per day for an adult, and it is 1 to several days or months as an administration interval. It is preferable to administer once.

The contents of all patents and references that are expressly cited herein are hereby incorporated by reference as part of this specification. In addition, all the content described in the specification and drawings of Japanese Patent Application No. 2003-330258, which is the basis of priority claims of the present application, is incorporated herein as part of this specification.

EXAMPLE

The present invention will be described in more detail by way of examples. However, these Examples are shown by way of example in order to demonstrate this invention more concretely, This invention is not limited by the following Example.

In addition, the abbreviation of the amino acid used in the following example is as follows.

A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, P is proline, R Is arginine, S is serine, T is threonine, V is valine, and Y is tyrosine.

Example 1 Detection of IgG Reactive with HCV Peptides in Serum of HCV Infected Patients

Peptide

Synthetic peptides with HLA-A2 binding motif or HLA-A24 binding motif from the conserved region of HCV genotype 1b protein were used (Table 1). As a negative control, an HIV derived peptide with an HLA-A2 binding motif was used. All of these peptides were commercially available products, and their purity was 90% or more when analyzed by reversed phase high pressure liquid chromatography.

The cutoff value is 1.83.

No. 40 Positive rate HCV Infectious Diseases 60 56 (93.3%) Hepatitis B virus infected 24 0 (0%) Hepatitis B Virus Vaccine 10 0 (0%) A healthy person 27 0 (0%) Autoimmune patients 22 0 (0%) HTLV-1 infected 10 8 (80%) HIV infected 3 3 (100%) count 156 67 C-35 Detection rate 93.3% HCV specificity 88.5% OD rate P <0.001

The detection rate was calculated as the number of patients positive at the cutoff value (mean ± 3SD).

NS5A-2132 is 0.202 and C-35 is 0.13.

Statistical analysis was performed by the χ ² test.

ELISA

Peptide specific IgG in serum was measured by ELISA. First, each peptide was dissolved in dimethyl sulfoxide (DMSO) and stored at -80 ° C. Peptides were diluted with 0.1 M sodium carbonate / sodium bicarbonate solution containing disuccinimidylsuberate (DSS) as a cross linker. The ELISA plates were allowed to react by 20 μg / well peptide at 4 ° C. overnight. The wells were washed three times with 0.05% Tween20-PBS (PBST) and the plates were blocked with Block Ace® and left overnight at 4 ° C. Plasma or serum samples were diluted 100-fold, 200-fold and 400-fold with 0.05% Tween20-Block Ace, respectively, and the resulting samples were added 100 μl per well. After incubating the sample for 2 hours at 37 ° C, the plate was washed 9 times with PBST, and 100 µl of each 1000-fold diluted rabbit anti-human IgG (γ-chain specific) rule was added and incubated at 37 ° C for 2 hours. After washing nine times with PBST, 100 μl of horseradish peroxidase dextran polymer covalently bound to mouse anti-rabbit IgG diluted 100-fold was added per well, and the plate was incubated at room temperature for 40 minutes. After washing the plate, 100 μl of tetramethylbenzidine substrate solution was added to each well, and 2.0 M phosphoric acid was added to stop the reaction. The cutoff value was computed as the average +/- 3SD of the light density control of a healthy person.

Peptide specific IgG of  Absorption and elution

100 μl of plasma or serum sample per well was diluted with 0.05% Tween20-Block Ace® and absorbed at 37 ° C. for 2 hours in peptides (20 μg per well) immobilized in the wells of the plate. After this procedure was repeated three times, peptide specific IgG levels in the supernatants were measured by ELISA. Antibodies bound to peptide immobilization plates by initial adsorption were neutralized with 30 μl of 5 M NaCl / 50 mM citrate buffer (pH3.0) (0.05% Tween20-Block Ace (pH7.5) of 1.0 M Tris-HCl) per well. Was eluted and the peptide specific IgG levels in the eluted fractions were measured by ELISA.

Peptide specific CTL  analysis

The method used for detection of peptide specific CTL is a method commonly used in the art. PBMCs were placed in each well of a U bottom 96-well microculture plate to be 1 × 10 ⁵ cells per well and incubated in 200 μl of medium with 10 μM of peptide. The composition of this medium was 45% RPMI-1640, 45% AIM-V®, 10% fetal bovine serum (FCS), 100 U / ml of interleucine 2 (IL-2), and a 1 mM MEM ratio. It consists of an essential amino acid solution. At 3, 6, and 9 days of culture, except for half the medium, each was replaced with fresh medium containing the corresponding peptide (20 μg / ml). On the 12th day of culture, the cultured cells were harvested and tested for the ability to produce interferon gamma (IFN-γ) in response to T2 or CIR-A2402 cells previously attached to either the corresponding peptide or HIV peptide as a negative control. Four wells were used for each peptide and analysis was performed twice. Background IFN-γ production in response to HIV peptide was subtracted from the data obtained. Inhibition assays showed that peptide-reactive CTLs were purified using a CD8 isolation kit, and peptide-specific IFN-γ production was determined using 20 μg / ml anti-HLA class I (W6 / 32, IgG2a) monoclonal antibody, anti-CD8 (Nu). -Ts / c, IgG2a) monoclonal antibodies or anti-HLA class II (H-DR-1, IgG2a) monoclonal antibodies were measured in the presence of either. Background IFN-γ production was subtracted from the data values obtained.

statistics

The numerical value is shown as mean +/- SD. Statistical analysis was performed using the Mann-Whitney U test and the chi-square test. P <0.05 values are considered statistically significant.

HCV  Detection of Antibodies Against Peptides

The serum levels of 12 HCV infection patients and 10 healthy (HD) sera against 62 peptides were measured (Table 1). The positive rate of the antibody with respect to each peptide is shown in Table 1. As a result, a plurality of peptides were found that reacted with IgG in the blood of HCV-infected patients at a high rate and hardly reacted with healthy serum.

For example, NS5A-2132 of No 3 (corresponding to 2132 to 2140 of NS5A protein of HCV, the same is omitted below), No 11 (NS3-1266), No 12 (E2-488), No 13 (E1 -213), No 22 (NS2-947), No 25 (NS3-1081), No 32 (C-176), No 38 (C-173), No 40 (C-35), No 62 (NS5B-2990 ), And the like. The results of measuring IgG in blood of HCV-infected patients by typical ELISA are shown in FIGS. 1 and 2.

In the patient serum shown in FIG. 1, IgG antibody against C-35 was detected.

Among the four patient sera shown in FIGS. 2A-D, NS5A-2132 (A), NS3-1081 (B), E2-488 and E1-213 (C), C-176 and C-173, respectively. (D) Reactive IgG antibodies were detected The peptide specificity of these peptide reactive antibodies was confirmed by absorption and elution tests.

The absorption and elution test results of the C-35 peptide reactive antibody in the patient serum shown in FIG. 1 are shown in FIG. 3 as a representative example. This antibody was taken up by C-35 but not by the unrelated peptide NS5A-2252 (top of FIG. 3). This antibody was also eluted from the C-35 adsorption fraction (bottom of FIG. 3).

Among the Table 1, the C-35 peptide-reactive antibody was 100% in HCV infected persons and 0% in healthy subjects, showing high HCV specificity. The sequence of the C-35 peptide of HCV is partly related to several viruses, for example the tet protein of HIV-1 (amino acid sequence 54-58) or the env protein (amino acid sequence of 5-9, 158-162, 717-727). Homology is shown as. It also showed homology with several proteins of HTLV-I (pol 724-755, rex 5-9, rex 310-313, tax 70-74, etc.). Therefore, C-35 peptide-reactive antibody was used in 60 cases of HCV infection-related diseases (22 chronic hepatitis C, 20 cirrhosis, 18 liver cancers), 24 hepatitis B virus infected patients, 10 hepatitis B vaccine vaccinators, and health Twenty-seven cases of phosphorus, 22 patients with autoimmune disease, 10 patients with HTLV-I infection, 3 patients with HIV infection, and 156 cases with total HIV were examined by double blinding. The results are shown in Table 2. In these results, HCV specificity and detection rate of C-35 peptide reactive antibody were 88.5% and 93.3%, respectively.

Peptide specific CTL  Induction of activity

Six HLA-A24 binding peptides (No 3, 12, 13, 25, 32, 38) and 18 HLA-A2 binding peptides (No 40 to No 57) were infected with HLA-A24 or HLA-A2 positive HCV infection. Each of them was incubated with peripheral blood monocytes (PBMC). As a control, the PBMCs were incubated with HIV peptides. Then, regarding their cultured PBMCs, the ability of IFN-γ production to respond to C1R-A2402 cells or T2 cells pulsed with the corresponding peptide was examined. As a result, six HLA-A24 binding peptides (No 3, 12, 13, 25, 32, 38) were able to induce CTL in PBMC of 10 HLA-A24 positive HCV patients (FIG. 4). In addition, CTLs could be induced in PBMCs of 5 HLA-A2 positive HCV patients by HCV-derived HLA-A2 binding peptides (No 40 to No 57) (FIG. 5).

In addition, the cytotoxic activity of the peptide-stimulated PBMC was confirmed by the ⁵¹ Cr glass test. The typical result is shown to FIG. 6 and FIG.

Figure 6 shows cytotoxic activity of CTL induced in HLA-A24 positive HCV patient PBMC by HLA-A24 binding peptide (No 3, 12, 13, 25, 32, 38) derived from HCV. Cytotoxic activity against C1R-A2402 cells (labeled C1RA2402 in the figure) to which the inducing peptide was attached was significantly higher than C1R-A2402 cells (labeled to C1RA2402HIV in the figure) to which the HIV peptide of the control was attached.

7 shows the cytotoxic activity of CTL induced in HLA-A2 positive HCV patient peripheral blood PBMC by HCLA-derived HLA-A2 binding peptide (No 40, 41). The cytotoxic effect on T2 cells (indicated by T2 in the figure) to which the inducing peptide was attached was significantly higher than T2 cells (indicated by T2 HIV in the figure) to which the HIV peptide of control was attached.

As a result, these PBMCs exhibited significantly different levels of cytotoxicity against C1R-A2402 and T2 cells preloaded with the corresponding peptides (other than HIV peptides as negative controls).

This HLA class I restriction of cytotoxicity was confirmed by inhibition test using anti-CD8 monoclonal antibody (FIG. 8). 8 shows representative examples of inhibition by anti-CD8 antibodies of CTL activity. The cytotoxic activity of CTL induced in HLA-A2-positive HCV patient PBMC by HCV-derived HLA-A2 binding peptide (No 40) was inhibited by anti-CD8 antibody, but not by anti-CD4 or control anti-CD14 antibody. Did.

Example 2 Detection of IgG Responsive to HCV Peptides in Serum of HLA- A24 Positive HCV Infected Patients

Subject

More broadly, in HLA-A24 positive HCV infected patients, IgG that was reactive with HCV peptide was detected. Sixty HCV1b ⁺ patients were judged by a second or third generation immunoassay test and were cellopositive with respect to anti HCV antibody (Ab). The genotype of HCV in the serum of the patient was determined by RT-PCR. Diagnosis of the patients at the time of serum sampling was made by biochemical analysis, ultrasonography, computed tomography, and histological findings. The diagnostic results are as follows: chronic hepatitis (CH, n = 24), cirrhosis (LC, n = 18), and hepatocellular carcinoma (HCC, n = 18). As a negative control, serum samples from 20 healthy donors (HD) with normal hepatic function and no history of hepatitis virus were used.

Measurement of Peptides and Peptide Reactive Antibodies

44 synthetic peptides were used, derived from regions well conserved of HCV1b protein and selected based on high binding scores with HLA-A24 molecules (Bioinformatics and Molecular Analysis Section, NIH, Bethesda, MD). As a negative control, an HIV-derived peptide (RYLRDQQLLGI (SEQ ID NO: 63)) having an HLA-A24 binding motif was used. Binding scores and sequences are shown in Table 3. For screening on the reactivity of peptides to serum IgG, their desalting grade (> 70%) peptides were purchased from BioSyntehsis (Lewisville, TX) or SynPep (Dublin, Calif.). In subsequent experiments, purified peptide (> 90%) was used. Plasma levels of peptide specific IgG were measured by enzyme antibody method (ELISA). Specificity testing of anti-peptide IgG in plasma samples was performed by absorption and elution of peptide reactive IgG.

Peptide specific CTL  analysis

The method used for detection of peptide specific CTL precursor cells is as described in Hida et al. (Cancer Immunol Immunother 2002; 51: 219-228). For the inhibition test, 20 μg / ml of anti HLA class I (W6 / 32, IgG2a), anti CD8 (Nu-Ts / c, IgG2a), anti CD4 (Nu-Th / i, IgG1b), and anti HLA-class II (H-DR-1, IgG) monoclonal antibodies were used.

NS5A  By gene Transfected  Peptide Stimulation on Cells Of PBMC CTL  activation

The cDNAs of NS5A, HLA-A2402, and HLA-A2601 were obtained by RT-PCR from Huh7 (NNU50-1) cells, C1R-A2402 cells, and KE4-CTL cells, respectively, and the expression vector pCR3.1 (Invitrogen, San Diego , CA). Huh7 (NNU50-1) cell line is a replicon cell derived from HCV1b infected cell line and expresses the protein of NS5B from NS3 (Kishine et al., Biochem Biophys Res Commun 2002; 293: 993-999). Subsequently, 100 ng of NS5A, HLA-A2402 or NS5A, and HLA-A2601cDNA (as negative control) were mixed in 100 μl of Opti-MEM (Invitrogen) containing 0.6 μl of Fugene6 (Roche Molecular Biochemicals, Indianapolis, IN). And incubated for 30 minutes. Next, 100 μl of the mixture was added to COS-7 cells (1 × 10 ⁴ cells), which was incubated for 6 hours. 100 μl of RPMI-1640 medium containing 20% FCS was added to the mixture, and COS-7 cells were incubated for 2 days to add NS5A 2132-2140 stimulated PBMC (2 × 10 ⁵ cells / well). After incubation for 18 hours, 100 μl of supernatant was removed and IFN-γ concentration was measured by ELISA in a 3-well assay. To construct a stable transfectant cell line, either pCR3.1-NS5A or pCR3.1-HLA-A3101 (negative control) was transferred to C1R-A2402 cells using Gene Pulser (BioRAD, Richmond, CA). Transfection was performed by electroporation. These cells were incubated for 30-40 days in the presence of genetesin (G418) to recover genetesin resistant cells. Expression of the NS5A protein in the transfectant was done by Western blot analysis using anti NS5 polyclonal antibodies (Abcam Limited, Cambridge, UK).

Proliferation inhibition and antibody dependent cellular cellular injury ( ADCC )

Huh7 cell lines were incubated 24, 48, and 72 hours in the presence of several sera in wells of flat 96-well microculture plates. After incubation, Huh7 cells were counted with Cell Counting Kit-8 (10 μl / well) (Dojindo, Japan). For ADCC analysis, freshly isolated PBMCs from HLA-A24 positive HD with 10% FCS and RPMI-1640 medium containing non-functionalized serum (56 ° C., 30 min) from several patients or HD (as negative control). 1.5 hours preincubation. C1R-A2402 cells and C1R cells (as negative controls) were incubated with NS5A 2132-2140 peptide (10 μM) for 2 hours, radiolabeled with Na ₂ ⁵¹ CrO ₄ for 1.5 hours, washed and used as target cells. Peptide stimulated PBMCs were incubated with target cells at 40, 20, and 10 to 1 effector to target cell (E / T) ratios in wells of a 96 U bottom microculture plate. After 6 hours incubation at 37 ° C., the acellular supernatant was recovered to determine ADCC activity (Shomura, et al., Br J Cancer 2004; 90: 1563-1571).

statistics

Statistical analysis was performed using the Student's t test and the Mann Whitney U test. The value of P <0.05 was considered statistically significant.

HCV For peptides Liquid  Detection of responses

To screen the humoral response to peptides, the levels of each IgG reactivity against 44 peptides were measured in the plasma of 12 patients infected with HCV-1b. Ten HD plasmas were used as negative controls. The level of peptide reactive IgG was measured by ELISA with respect to serially diluted plasma samples, and the absorbance (OD) value of each sample was shown. The cutoff values of these peptides at a 1: 100 plasma dilution were 0.18 OD {mean (0.08) + 2SD (0.05 × 2) of 10 HD's. IgG reactivity at significant levels (> 0.18 OD at a serum dilution of 1: 100) for NS5A-2132, C-176, E2-488, E1-213, C-173, and NS3-1081 peptides was observed in 12 patients. In plasma, detection was possible in 12, 11, 10, 9, 8, and 5 patients, respectively (FIG. 9). As expected, these peptides were not all responsive to plasma from 10 HD's. The reactivity of peptides to peptide specific IgG is summarized in Table 3.

a) Levels of peptide specific IgG in serum of 12 HCV-1b ⁺ patients were measured by ELISA.

The cutoff value was determined to be 0.18.

b) Binding scores of peptides to HLA-A24 molecules were retrieved using the Bioinformatics and Molecular Analysis Section, NIH, Bethesda, MD (http://bimas.dcrt.nih.gov/molbio/hla_bind/).

Each of the six peptides described above, and a negative control peptide having a purity of at least 90%, was tested for 60 HCV1b ⁺ patients (CH, n = 24; LC, n = 18; and HCC, n = 18) and 20 HD. The reactivity with respect to each serum sample from the serum of the test was tested (Fig. 10).

For each of these samples a value of 100: 1 dilution was plotted. Mean ± SD of IgG (OD value) was as follows: NS5A-2132 (0.48 ± 0.36), E2-488 (0.18 ± 0.19), E1-213 (0.10 ± 0.21), NS3-1081 (0.036 ± 0.14), C-176 (0.09 ± 0.14) and C-173 (0.04 ± 0.13). * Indicates P <0.05 by Mann Whitney U test. The average level of IgG reactive for NS5A-2132, E2-488, E1-213, or C-173 was statistically significantly higher than that of HD, but not for NS3-1081 or C-176. . IgG (> 0.101 OD in serum dilution of 1: 100) exhibiting significant levels of reactivity against the NS5A-2132, E2-488, E1-213, C-173, NS3-10810, and C-176 peptides, Detectable in 57 (95%), 44 (73%), 28 (47%), 23 (38%), 21 (35%), and 17 (28%), respectively, of the plasma of 60 patients tested there was. On the other hand, all 20 HD serums could not detect significant level of IgG (FIG. 10).

Next, the peptide specificity of IgG which has reactivity with each of these peptides was confirmed by the absorption and elution test. Each serum sample was absorbed three times at 37 ° C. in either the corresponding peptide or unrelated peptide (HIV; used as negative control) and peptide specific IgG was measured by ELISA (FIG. 11). In the dissolution test, the IgG molecules bound to the peptide immobilization plates after the first absorption were eluted with the corresponding peptide or irrelevant peptide, and then the level of peptide specific IgG in the elution fraction was measured by ELISA. The mean ± SD of OD values of three measurements is shown for a representative result (FIG. 12). * Indicates P <0.05 by two-tailed Student's t test.

As expected, each IgG for all six peptides was absorbed by the corresponding peptide, but not by an irrelevant peptide (HIV peptide). This IgG was eluted from the binding fraction by the corresponding peptide, but not by an unrelated peptide. Taken together, these results suggest that IgG that is reactive to each peptide is specific for HCV1b derived peptides.

Induction of Peptide Specific Cellular Responses

PBMCs from HLA-A24 positive patients (n = 12, 6 CH, 3 LCs, 3 HCCs) infected with HCV1b and HLA-A24 positive HDs (n = 5) uninfected with HCV In addition, IFN production in response to C1R-A2402 cells pulsed with the corresponding peptide was incubated for 13 days with each of six peptides (purity> 90%) or a control HIV peptide. PBMCs from patients (n = 12) and HD (n = 5) of HLA-A24 positive HCV1b ⁺ were stimulated with four wells (15 × 10 ⁴ / well) in six peptides. On day 14 of the culture, peptide stimulation PBMCs (80-120 × 10 ⁴ / well) from each well were collected separately and divided into four equally. Two of them were tested separately for the ability to generate IFN-γ in response to C1R-A2402 cells pulsed with the corresponding peptide. The other two were tested with cells using negative control peptides (HIV). IFN- [gamma] generated in response to HIV peptide (<50 pg / ml) was subtracted in the background. * Indicates P <0.05 by two-tailed Student's t test.

HCV peptides of C-173, C-176, E1-213, E2-488, NS3-1081, and NS5A-2132 were 1, 3, 4, 6, 2, and 7 of 12 patients, respectively (FIG. 13, 14), and from 0, 0, 1, 1, 1, and 1 of 5 HD, respectively, induced significant levels (p <0.05) of IFN-γ production (data not shown). Of these six peptides, the NS5A-2132 peptide was recognized by cellular and humoral immunity in PBMCs from 7 of 12 patients and 57 sera of 60 HCV1b ⁺ patients tested.

Cytotoxicity of these peptide-stimulated PBMCs was confirmed by 6 hours of ⁵¹ Cr glass analysis (FIG. 15). Peptide-stimulated PBMCs showing a positive response by IFN- [gamma] production analysis (described above) were grown by incubation with IL-2 only in vitro. Next, cytotoxicity against C1R-A2402 cells pulsed with the corresponding peptide or HIV peptide (negative control) was tested at three different E / T cell ratios by a standard 6 hour ⁵¹ Cr glass assay. Representative results are shown. Values are expressed as mean ± SD of% of specific lysis. * Indicates P <0.05 by two-tailed Student's t test. PBMCs stimulated with NS5A-2132, E2-488, or E1-213 peptides showed significant levels of cytotoxicity against C1R-A2402 cells preloaded with the corresponding peptide, but not with HIV peptides (FIG. 15). ).

Peptide stimulation PBMCs used in the experiments shown in Fig. 15 were 20 μg / ml of anti-HLA-class I (W6 / 32, IgG2a), anti-HLA-class II (H-DR-1, IgG2a), and anti-CD8 (Nu- In the presence of Ts / c, IgG2a), and anti CD4 (Nu-Th / i, IgG1) monoclonal antibodies (mAb), cytotoxicity was tested for C1R-A2402 cells pulsed with the corresponding peptide. Anti CD14 (JML-H14, IgG2a) mAb was used as negative control. Six hours of ⁵¹ Cr glass analysis were performed at three different E / T ratios. Values are expressed as mean ± SD of% of specific injury (FIG. 16). * Indicates P <0.05 by two-tailed Student's t test.

Cytotoxicity against C1R-A2402 cells pulsed into each of the three peptides was inhibited by anti-HLA-class I (W6 / 32) or CD8 monoclonal antibody (mAb), but none of the other mAbs tested It was not inhibited. This indicates that peptide specific cytotoxicity is mediated by HLA-Class I binding, mainly by CD8 ⁺ T cells (FIG. 16). In contrast, such cytotoxicity could not be detected in PBMCs stimulated with the remaining three peptides (C-173, C-176, or NS3-1081) (data not shown).

Next, by using COS-7 cells transiently transfected with NS5A and HLA-A2401 genes as target cells, it was examined whether the NS5A-2132 peptide-stimulated PBMC recognized a naturally occurring peptide. As a negative control, COS-7 cells transiently transfected with NS5A and HLA-A2601 genes were used. NS5A-2132 Peptide Stimulation PBMCs from Patients # 1 and # 2 Recognize COS-7 Cells Transientally Transfected with NS5A and HLA-A2401 Genes as Target Cells to Test IFN-γ Activity It was. As a negative control, COS-7 cells transiently transfected with NS5A and HLA-A2601 genes were used. Values represent mean ± SD of% of IFN-γ production in three assays at E / T ratio 20: 1 (FIG. 17). * Indicates P <0.05 by two-tailed Student's t test.

As expected, peptide stimulated PBMCs from patients # 1 and # 2 showing CTL activity (Figure 13) recognized COS-7 cells transfected with NS5A and HLA-A2401 genes, resulting in significant amounts of IFN -γ was generated (FIG. 17). In contrast, these PBMCs did not respond to COS-7 cells with NS5A and HLA-A2601 genes used as negative controls. In addition, peptide-stimulated PBMCs from two other patients (# 3 and # 4) that did not exhibit CTL activity also showed significant amounts of IFN by recognition of COS-7 cells transfected with NS5A and HLA-A2401 genes. No -γ was generated (data not shown).

Next, cytotoxicity was tested for C1R-A2402 cells stably transfected with NS5A gene and used as target cells. Expression of HLA-A24 molecule in C1R-A2402 cells stably transfected with NS5A gene and expression of HLA-A31 molecule in C1R-A2402 cells stably transfected with HLA-A31 gene (negative control) , Confirmed by flow cytometry analysis using FACScan (FIG. 18).

Next, cytotoxicity using the C1R-A2402 cells stably transfected with the NS5A gene as target cells was tested by 6 hours of ⁵¹ Cr glass analysis. C1R-A2402 cells stably transfected with a negative control gene (HLA-A3101) were used as negative controls and C1R-A2402 cells pulsed with NS5A-2132 peptide were used as positive controls. Six hours of ⁵¹ Cr glass analysis were performed at three different E / T ratios. Values are expressed as mean ± SD of% of specific lysis. * Indicates P <0.05 by two-tailed Student's t test. PBMCs stimulated with the NS5A-2132 peptide did not transfect pre-pulsed with C1R-A2402 cells transfected with the NS5A gene, and the corresponding peptide, compared to cytotoxicity against C1R-A2402 cells transfected with the negative control gene. For both C1R-A2402 cells, higher levels of cytotoxicity were shown (FIG. 19). These results suggest that NS5A-2132 peptide stimulating PBMCs readily recognized naturally occurring peptides on HLA-A2402 molecules in HCV1b ⁺ cells.

term NS5A -2132 IgG  Further research on

To better understand the potential role of anti-peptide IgG, uptake and elution assays examined whether anti-NS5A-2132 IgG recognizes the entire NS5 protein. NS5A-2132 and HIV peptides were used as positive and negative controls, respectively (see above for details of absorption and elution tests). As a result, the anti-peptide IgG was neither absorbed nor eluted with all NS5 proteins (FIG. 20). This suggests that this peptide IgG did not react with the entire NS5 protein.

Next, Huh7 cells were incubated for 3 days in the presence of patient serum (serum from two HCV1b ⁺ patients whose serum had high levels of anti NS5A-2132 activity). As a negative control serum and FCS from two HDs were used. The number of viable Huh7 (NNU50-1) cells is counted with Cell Counting Kit 8 (10 μl / well) to represent the average of three assays. All of the tested sera did not inhibit the proliferation of Huh7 (NNU50-1) cells under their culture conditions (FIG. 21). In addition, it was investigated whether anti-NS5A-2132 IgG has the ability to mediate ADCC activity. PBMCs isolated from HLA-A24 positive HD were tested for cytotoxicity against C1R-A2402 cells previously pulsed with this peptide in the presence of the four nonfunctionalized sera described above. However, none of the serum tested under these conditions showed ADCC activity (FIG. 22).

Example  3. HCV  Prediction of Prognosis of Infected Person

Subject

Serum was obtained from 33 patients with HCV related disease in the cohort study of 1995-2002. The results of the follow up survey of 33 patients are shown in Table 4. Serum levels of patients with chronic hepatitis (CH, n = 68), cirrhosis (LC, n = 43), and hepatocellular carcinoma (HCC, n = 52) were also used for the analysis. These patients were diagnosed at the first serum sampling by biochemical and histological findings, ultrasonography, and computed tomography. Anti-HCV antibodies were obtained by chemiluminescent enzyme immunoassay (CLEIA) kit (Lumipulse II HCV, Fujirebio Inc., Tokyo, Japan) or by second or third generation enzyme immunoassay assays (SRL, Tokyo, Japan). Measured. HCV-RNA in serum was detected using RT-PCR (SRL, Tokyo, Japan). HCV genotype was determined by direct sequencing of HCV in the serum of patients with RT-PCR (SRL, Tokyo, Japan). Serum was also obtained from a subject not infected with HCV. This included 24 subjects with autoimmune diseases (6 patients with systemic lupus erythematosus, 1 patient with Behcet's disease and 17 patients with atopic dermatitis), and 17 cases of hepatitis B virus (HBV) infection ( Positive to HBV surface antigen), 3 patients with human immunodeficiency virus (HIV), and 10 cases with human T cell leukemia virus type I (HTLV-1) infection. As a negative control, sera from 37 people with normal hepatic function without a history of hepatitis virus or vaccination of HBV were tested.

CH, chronic hepatitis; LC, cirrhosis; HCC, liver cancer; ASC, Asymptomatic Health Carrier

Peptide

The following two peptides with a purity of 90% or higher were purchased from BIOSYNTHESIS (Lewisville, TX); HCV1b core protein 35-44 (YLLPRRGPRL (SEQ ID NO: 1); can induce HLA-A2 binding CTL activity), and HCV1bNS5A protein 2132-2140 (RYAPACKPL (SEQ ID NO: 2); induce HLA-A24 binding CTL activity Can be). As a negative control, an HIV derived peptide having an HLA-A2 binding motif (SLYNTVATL (SEQ ID NO: 64)) and an HLA-A24 binding motif (RYLRDQQLLGI (SEQ ID NO: 63)) was used.

Measurement of Antibodies Responsive to Peptides

The level of peptide specific IgG was measured by enzyme antibody method (ELISA). Briefly, each peptide was dissolved in dimethylsulfoxide (DMSO) and stored at -20 ° C. Peptides (20 μg / well) diluted with 0.1 M carbonate buffer containing the chemical crosslinking agent disuccinimidylsuberate (DSS) (PIERCE, Rockford, IL) were bound to ELISA plates. The wells were washed three times with 0.05% Tween20-PBS (PBST). Plates were blocked overnight at 4 ° C. with Block Ace (Yukijirushi, Tokyo, Japan). Serum samples were diluted 100-fold, 200-fold, 400-fold with 0.05% Tween20-Block Ace and 100 μl / well of sample was added to each well. After 2 hours incubation at 37 ° C., the plates were washed 9 times with PBST, and at 37 ° C. with rabbit anti human IgG (γ chain specific) (DAKO, Glostrup, Denmark) diluted 100 μl / well of 1000-fold. Time incubated. After 9 washes, 100 μl / well of 1: 100 diluted anti rabbit IgG binding horseradish peroxidase dextran polymer (En Vision, DAKO) was added to each well and the plate was incubated at room temperature for 40 minutes. . After washing, 100 μl / well of tetramethylbenzidine substrate solution (KPL, Guildford, UK) was added and the reaction was stopped by adding 1.0 M phosphoric acid.

statistics

Statistical analysis was done using the χ ² test. The value of p <0.05 was considered statistically significant.

Cross reactive, HLA  Binding, and Geno type  Binding

The cross reactivity of two antibodies with reactivity to HCV1b derived peptides was investigated. Serum from 60 HCV patients and patients not included in the cohort study (CH, n = 22, LC, n = 21, and HCC, n = 17) was taken from 35-44 (C-35) and NS5A of the core. The reactivity of 2132-2140 (NS5A-2132) to the peptide was investigated. Serum was also obtained from 24 subjects with autoimmune diseases, 17 cases of HBV infection and 10 cases of HTLV-1 infection. Serum from 37 HDs was used as a negative control. The level of peptide reactive IgG in serially diluted serum samples was measured by ELISA. The value of the absorbance (OD) of each sample was shown. Representative results of OD at serum dilution of 100: 1 are shown. The cutoff value was set to 0.093 (mean + 2SD of OD from HD). Statistical analysis was performed using the χ ² test. The value of p <0.05 was considered statistically significant.

As a result, significant levels of anti-C-35 IgG were detected in 56/60 (93.3%) of HCV positive patients, and there was no significant difference in IgG levels between groups 3 (CH, LC, and HCC patients) ( Figure 23). Except for HTLV-1 patients, serum from other groups was not positive for anti-C-35 antibodies. In the serum of 8/10 cases of HTLV-1 + subjects, a low but significant level of anti-C-35 IgG was detected. Significant levels of anti NS5A-2132 IgG were detected in 45/60 (75%) of patients. IgG levels were high in CH patients, moderate in LC, and low in HCC patients between groups 3 (p <0.05 vs CH) (FIG. 23). As for serum from other groups, anti-NS5A-2132 IgG was positive in most of the cases tested. In addition, 3/37 HD serum had positive anti-NS5A-2132 IgG.

Next, in 29 patients with HCV related diseases, the correlation between the HLA-class IA phenotype and the level of anti C-35 IgG or anti NS5A-2132 IgG was examined. HLA-class IA phenotype was determined by standard serological methods. Nine patients were HLA-A2 positive, 13 patients were A24 positive, and the remaining seven patients were A2 negative and A24 negative. Anti C-35 and anti NA5A 2132 were determined by standard ELISA and depicted OD values at 100: 1 serum dilution of each patient. All antibodies were detected in the majority of patients with HCV infection, regardless of the difference in HLA-Class I A phenotype (FIG. 24).

Next, in patients with HCV-1b (n = 29), HCV-2a (n = 16) and HCV-2b infection (n = 3), HCV genotype and anti-C-35 IgG or anti-NS5A- The correlation between the levels of 2132 IgG was examined by double blinding. HCV genotypes were determined by direct sequencing of HCV in the serum of patients. Results at 100: 1 dilution of all subjects are shown in FIG. 25. Anti C-35 and anti NA5A 2132 were measured by standard ELISA and depicted OD values at 100: 1 serum dilution of each patient. The cutoff value was set to 0.093 (mean + 2SD of OD from HD).

Anti-C-35 antibodies were detected in 26/30 HCV-1b, 15/15 HCV-2a, and 3/3 HCV-2b infected patients, respectively. Similarly, anti NS5A-2132 antibodies were detected in serum from HVC1b at 23/30, HCV-2a at 10/16, and HCV-2b infected at 3/3, respectively (FIG. 25). These results indicate that in HCV positive patients, both anti-C-35 antibody and anti-NS5A-2132 antibody were detected regardless of the difference between the HLA-class IA subtype and the HCV genotype.

The above results suggest that the level of anti-NS5A-2132 antibody correlates with the prognosis of HCV infected individuals, but not the anti-C-35 antibody.

Next, serum of CH (n = 24), LC (n = 22), HCC (n = 26), and HD (n = 9) was newly prepared, and the level of anti-NS5A-2132 IgG was measured (FIG. 26). ). The mean ± SD of CH, LC, HCC and HD groups was 0.51 ± 0.24, 0.27 ± 0.20, 0.26 ± 0.23, 0.08 ± 0.07, respectively. The levels of anti NS5A-2132 antibody in LC and HCC patients were significantly lower than in CH patients (p <0.05) but higher than HD. In order to compare these results with those measured by standard third generation assays, the levels of anti HCV antibodies were measured for all sera using commercially available kits (third generation assays, SRL). Levels are expressed as exponents (left column). The correlation between the anti HCV level and the anti NS5A-2132 level indicated by the exponent is shown in the right column. The levels of anti HCV antibodies measured by the third generation assay did not differ significantly between groups (CH; 10 ± 2.7, LC; 11 ± 1.4, HCC; 11 ± 1.1) (FIG. 27). In addition, HCV RNA levels were measured for these sera using commercially available kits (SRL, Japan). HCV RNA levels (360 ± 269.6) in HCC patients were lower (P <0.05) than CH patients (610 ± 347.3) or LC patients (610 ± 246.4) (FIG. 28). However, there was no obvious correlation between the levels of anti NS5A antibodies and HCV RNA levels (FIG. 28).

Cohort  Results of the study

In order to further investigate the correlation between anti-NS5A-2132 antibody and the prognosis of HCV-positive patients on an individual level, a sample from a cohort study conducted annually for residents infected with HCV, conducted from 1990 to 2002, 2 Serum levels of dog antibodies were examined. For this study, a total of 66 sera from 33 patients taken twice from the same subject in 1995 and 2002 were used. The diagnosis of these 33 patients at the time of 1995 was CH (n = 17), LC (n = 1), asymptomatic carrier (ASC) (n = 4), and past history of HCV infection (naturally recovered individuals). (n = 11) and at 2002 the history of CH (n = 13), LC (n = 4), HCC (n = 2), asymptomatic carrier (ASC) (n = 1), and HCV infection (n = 13). In other words, disease progression was not observed in 26 patients during 7 years, but progression was observed in the remaining 7 patients (Table 4). The OD values of anti-C-35 antibody and anti-NS5A-2132 IgG in each patient measured by serum dilution of 100: 1 in 1995 and 2002 are shown in FIG. 29. As expected, in most of 33 cases, the levels of anti-C-35 antibodies measured in 1995 were nearly identical to those measured in 2002, regardless of disease status. In contrast, the levels of anti-NS5A antibodies measured in 1995 decreased in all patients with advanced disease as measured in 2002. On the other hand, most of the 25 cases where the disease did not progress were almost identical to those measured in 2002. The median ± SD (0.67 ± 0.13) of the anti-NS5A-2132 antibody at the time of 1995 in the sera of 7 patients with advanced disease was significantly higher than the value measured in 2002 (0.27 ± 0.11) (p <0.05) .

Next, 33 subjects were divided into five groups (CH, LC, HCC, past history of HCV infection [recovered individuals], and ASC), two at 100: 1 serum dilution in 2002. The level of the antibody was plotted (FIG. 30). Statistical analysis was done using the χ ² test. The value of p <0.05 was considered statistically significant. The levels of anti-C-35 antibodies were all high in CH, LC, and HCC, and very low or undetectable in the cured individuals. On the other hand, the levels of anti NS5A-2132 antibodies were high in CH, recovered individuals and ASC, medium in LC, and lowest in HCC patients (p <0.05vsCH).

Example 4: Luminex (Luminex) method detection of IgG with HCV peptides with reactivity in the serum of HCV-infected patients by the HLA -A24 +

In the same manner as in Example 1, IgG having reactivity with HCV peptides in serum of HLA-A24 + HCV infected patients was detected using the Luminex method, which is considered to have higher sensitivity than the ELISA method.

Smile of peptide On the bead  For combine

Peptides were bound to microbeads (xMAP Multi-Analyte COOH Beads, manufactured by Luminex, Inc.), each having the content of each fluorescent dye identified by an identification code (hereinafter referred to as color code), as described below. 100 μl of color-coded uncoated microbeads were aspirated into each well of the filter plate, followed by a wash buffer (phosphate buffered saline (PBS) (pH 7.4 ± 0.1), Tween (trademark) 20 (0.05). % v / v)) twice to suck each well simultaneously. Next, 50 µl of 0.1 M MES (2-morpholinoethanesulfonic acid) buffer (pH 7.0) was added, and EDC (N-ethyl-N '-(3-dimethylaminopropyl) -carbodiimide hydrochloride) (1 mg / ml / 0.1 M MES buffer (pH 7.0)) was added in 10 μl each well. Several hundred μl of peptide (1 mg / ml, 0.1 M MES buffer, pH 7.0) were mixed with this washed microbead in each well. Thereafter, the microbeads mixed with the peptide were reacted in a dark place for 20 minutes at room temperature, and then additionally 10 μl of each well of EDC (1 mg / ml / 0.1M MES buffer (pH 7.0)) was added in a dark place. The operation of reacting at room temperature for 20 minutes was repeated twice. After aspirating the surplus, 1 M tris hydrochloric acid buffer (pH 7.0) was added to each 100 µl of each well, and reacted at room temperature for 15 minutes in a dark place. Next, the beads of each well were washed three times with the above washing buffer, and 0.05% sodium azide was added to the block ace (registered trademark) to recover the adjusted solution.

smile Bead Mix of  pharmacy

When preparing microbeads of each well, a bead solution obtained by binding peptides to color-coded microbeads as described above was added to have about 5,000 microbeads per well of the filter plate (about 1 μl of one bead per well). do. In addition, the bead mix was prepared by diluting the prepared microbeads in 10 equal amounts and diluting the total amount with the washing buffer (PBS, TWEEN® 20 (0.05% v / v)) to about 25 μl.

Preparation of Sample

Serum was used. Serum dilution diluted 100-1000 times with serum using reaction buffer (PBS (pH 7.4 ± 0.1), Tween® 20 (0.05% v / v), fetal bovine serum albumin (BSA) 10 mg / ml) 100 μl of each was prepared.

Anti Peptide Antibody Measurements

As a biotinylated secondary antibody, biotinylated goat anti human IgG (gamma chain specific) was used diluted in the reaction buffer. As the fluorescent dye-labeled streptovidin, 1 mg / ml of streptoavidine (SRPE) labeled with PE (phycoerythrin) was diluted (1/50 dilution) to 20 µl with the reaction buffer.

100 µl of the washing buffer was placed in each well of the filter plate, followed by suction removal. This washing operation was performed twice. Next, a 96 well filter plate containing 25 µl of the bead mix in each well was washed twice with the wash buffer. After washing, 100 μl each of the samples was added to all wells. Next, the filter plate was covered and shaken in a dark place using a plate shaker (300 rpm) at room temperature for 2 hours, followed by suction. Subsequently, 100 µl of the washing buffer was added to each well, followed by suction removal. This washing operation was performed three times.

Next, 100 μl of each biotinylated goat anti-human IgG (gamma chain specific) was put into each well as a biotinylated secondary antibody, and the filter plate was covered with a plate shaker (300 rpm) at room temperature for 1 hour in a dark place. Shaken. Thereafter, suction was carried out, and 100 µl of the washing buffer was added to each well to remove the suction. This washing operation was performed three times.

Subsequently, 100 µl of SRPE was put into each well, and the filter plate was covered and shaken with a plate shaker (300 rpm) at room temperature for 30 minutes in a dark place. Thereafter, suction was carried out, and 100 µl of the washing buffer was added to each well to remove the suction. This washing operation was performed three times. Thereafter, 100 µl of the washing buffer was put in each well, and shaken for 2 to 3 minutes using a plate shaker (300 rpm), and then 50 µl of the obtained sample was measured by a fluorescent flow meter system. The results are shown in FIGS. 31 and 32 and Table 5.

Example  5: using hepatitis C virus-derived peptide vaccine HCV  Treatment of Infection

In HCLA infected with HLA-A2 positive or HLA-A24 positive, the antiviral effect of the vaccine using the hepatitis C virus-derived peptide of the present invention was investigated. All the subjects were patients infected with HCV1b and did not respond to treatment with interferon + ribavirin. Hepatitis C virus-derived peptides C-35, NS5A-2132, E2-488, E1-213 and NS3-1081 were synthesized under GMP compliance, purified and stored as lyophilized powder. Peptides of C-35, NS5A-2132, E2-488 and NS3-1081 are dissolved in a small amount of DMSO (1 mg / 10-25 μl), and E1-213 is dissolved in 7% sodium bicarbonate injection solution (micron) (1 mg / 15 μl). They were each diluted (1-2 mg / ml) with physiological saline for injection and sterilized through a 0.22 μM filter. This solution was mixed with an equal amount of adjuvant (Montanide ISA-51) to obtain an emulsion injection. Among them, three HLA-A2 positive subjects received C-35 injection, and five HLA-A24 positive subjects received NS5A-2132 injection. Administration was performed by injecting into the flanks an emulsion containing 0.3 mg of peptide once per two weeks. The amount of HCV RNA and the values of GOT, GPT, γ-GTP, AFP were continuously monitored.

The results are shown in FIGS. 33 and 34. As can be seen from these figures, in most of the subjects tested, the amount of HCV RNA was significantly reduced after the start of vaccine administration, demonstrating that the peptide of the present invention was effective as an anti HCV vaccine.

The hepatitis C virus-derived peptide according to the present invention contains an HLA binding motif in its sequence, and is recognized by an antibody that responds to the hepatitis C virus, and further, HLA-A2 or HLA-A24 restrictive cytotoxicity. It has recognition by T cells.

Therefore, the hepatitis C virus-derived peptide of the present invention can be an effective vaccine against diseases caused by HCV infection.

<110> KURUME UNIVERSITY <120> HEPATITIS C VIRUS DERIVED PEPTIDES <130> PIK9002WO <150> JP 2003-330258 <151> 2003-09-22 <160> 64 <170> PatentIn version 3.1 <210> 1 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 1 Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu   1 5 10 <210> 2 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 2 Arg Tyr Ala Pro Ala Cys Lys Pro Leu   1 5 <210> 3 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 3 His Tyr Ala Pro Arg Pro Cys Gly Ile   1 5 <210> 4 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 4 Val Tyr Glu Ala Ala Asp Met Ile Met   1 5 <210> 5 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 5 Val Tyr His Gly Ala Gly Ser Lys Thr Leu   1 5 10 <210> 6 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 6 Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu   1 5 10 <210> 7 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 7 Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu   1 5 10 <210> 8 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 8 Glu Tyr Val Leu Leu Leu Phe Leu Leu   1 5 <210> 9 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 9 Met Tyr Thr Asn Val Asp Gln Asp Leu   1 5 <210> 10 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 10 Gln Tyr Leu Ala Gly Leu Ser Thr Leu   1 5 <210> 11 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 11 Leu Tyr Gly Val Trp Pro Leu Leu Leu   1 5 <210> 12 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 12 Ala Tyr Ser Gln Gln Thr Arg Gly Leu   1 5 <210> 13 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 13 His Tyr Lys Leu Phe Leu Ala Arg Leu   1 5 <210> 14 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 14 Thr Tyr Ala Thr Tyr Gly Lys Phe Leu   1 5 <210> 15 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 15 Ala Tyr Met Ser Lys Ala His Gly Ile   1 5 <210> 16 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 16 Pro Tyr Ile Glu Gln Gly Met Gln Leu   1 5 <210> 17 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 17 Asn Phe Ile Ser Gly Ile Gln Tyr Leu   1 5 <210> 18 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 18 Pro Tyr Phe Val Arg Ala His Gly Leu   1 5 <210> 19 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 19 Gln Phe Lys Gln Lys Ala Ile Gly Leu   1 5 <210> 20 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 20 Ile Phe Thr Ile Thr Lys Ile Leu Leu   1 5 <210> 21 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 21 Gly Tyr Ile Pro Ile Val Gly Ala Pro Leu   1 5 10 <210> 22 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 22 Met Tyr Val Gly Gly Val Glu His Arg Leu   1 5 10 <210> 23 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 23 His Tyr Val Gln Met Ala Leu Met Lys Leu   1 5 10 <210> 24 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 24 Thr Tyr Val Tyr Asp His Leu Thr Pro Leu   1 5 10 <210> 25 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 25 Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu   1 5 10 <210> 26 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 26 Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu   1 5 10 <210> 27 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 27 Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu   1 5 10 <210> 28 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 28 His Tyr Pro Cys Thr Val Asn Phe Thr Ile   1 5 10 <210> 29 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 29 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile   1 5 10 <210> 30 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 30 Thr Phe Leu Val Gly Leu Asn Gln Tyr Leu   1 5 10 <210> 31 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 31 Val Phe Val Gly Leu Ile Leu Leu Thr Leu   1 5 10 <210> 32 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 32 Glu Phe Trp Glu Ser Val Phe Thr Gly Leu   1 5 10 <210> 33 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 33 Leu Phe Leu Ala Arg Leu Ile Trp Trp Leu   1 5 10 <210> 34 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 34 Tyr Phe Ile Thr Arg Ala Glu Ala His Leu   1 5 10 <210> 35 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 35 Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu   1 5 10 <210> 36 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 36 Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu   1 5 10 <210> 37 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 37 Phe Phe Thr Glu Val Asp Gly Val Arg Leu   1 5 10 <210> 38 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 38 Ser Phe Ala Ile Lys Trp Glu Tyr Val Leu   1 5 10 <210> 39 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 39 Asp Leu Met Gly Tyr Ile Pro Leu Val   1 5 <210> 40 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 40 Leu Leu Ala Leu Leu Ser Cys Leu Thr Val   1 5 10 <210> 41 <211> 8 <212> PRT <213> Hepatitis C Virus <400> 41 Ile Leu His Thr Pro Gly Cys Val   1 5 <210> 42 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 42 Ser Met Val Gly Asn Trp Ala Lys Val   1 5 <210> 43 <211> 11 <212> PRT <213> Hepatitis C Virus <400> 43 Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn Val   1 5 10 <210> 44 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 44 Cys Ile Asn Gly Val Cys Trp Thr Val   1 5 <210> 45 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 45 Leu Leu Cys Pro Ala Gly His Ala Val   1 5 <210> 46 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 46 Lys Leu Val Ala Leu Gly Ile Asn Ala Val   1 5 10 <210> 47 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 47 Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr   1 5 10 <210> 48 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 48 Ser Leu Met Ala Phe Thr Ala Ala Val   1 5 <210> 49 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 49 Leu Leu Phe Asn Ile Leu Gly Gly Trp Val   1 5 10 <210> 50 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 50 Ile Leu Ala Gly Tyr Gly Ala Gly Val   1 5 <210> 51 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 51 Ile Leu Asp Ser Phe Asp Pro Leu Val   1 5 <210> 52 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 52 Arg Leu Ile Val Phe Pro Asp Leu Gly Val   1 5 10 <210> 53 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 53 Gly Leu Gln Asp Cys Thr Met Leu Val   1 5 <210> 54 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 54 Ala Leu Ala His Gly Val Arg Ala Leu   1 5 <210> 55 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 55 Asn Leu Pro Gly Cys Ser Phe Ser Ile   1 5 <210> 56 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 56 Ser Tyr Thr Trp Thr Gly Ala Leu Ile   1 5 <210> 57 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 57 His Tyr Arg Asp Val Leu Lys Glu Met   1 5 <210> 58 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 58 Trp Phe Met Trp Cys Leu Leu Leu Leu   1 5 <210> 59 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 59 Ala Phe Tyr Gly Val Trp Pro Leu Leu   1 5 <210> 60 <211> 9 <212> PRT <213> Hepatitis C Virus <400> 60 Gln Phe Lys Gln Lys Ala Leu Gly Leu   1 5 <210> 61 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 61 Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe   1 5 10 <210> 62 <211> 10 <212> PRT <213> Hepatitis C Virus <400> 62 Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu   1 5 10 <210> 63 <211> 11 <212> PRT <213> Human Immunodeficiency Virus <400> 63 Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile   1 5 10 <210> 64 <211> 9 <212> PRT <213> Human Immunodeficiency Virus <400> 64 Ser Leu Tyr Asn Thr Val Ala Thr Leu   1 5  

Claims (2)

A pharmaceutical composition for diagnosing, preventing, treating or predicting prognosis of hepatitis C virus infection, comprising as an active ingredient a hepatitis C virus-derived peptide comprising SEQ ID NO: 1. A kit for diagnosing or preventing hepatitis C virus infection, comprising the pharmaceutical composition according to claim 1.

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